2型猪链球菌强毒株与无毒株胞外蛋白的双向电泳图谱比较分析

利用双向电泳技术可以对体外培养的2型猪链球菌强毒株和无毒株进行胞外蛋白质组比较,寻找与毒力相关的蛋白质.2型猪链球菌强毒株和无毒株在无蛋白细菌培养液中37℃摇床培养16 h,从培养物上清中获得胞外蛋白.第一向电泳用pH4-7线性IPG胶条进行等电聚焦,第二向电泳用SDS-PAGE凝胶再分离蛋白,经过考马斯亮蓝R350染色,图像扫描后,利用软件分析处理图像.在2型猪链球菌强毒株和无毒株的胞外蛋白质图谱中都分别检测出180±10个蛋白质斑点,它们的蛋白质分子质量分布基本相似;在所检测到的差异蛋白质斑点中,其中有50个蛋白斑点只存在于无毒株中而强毒株中不存在,有52个蛋白斑点只存在于强毒株中而无毒株中不存在,有7个蛋白斑点的表达量相差达5倍以上.这为研究2型猪链球菌的致病机理提供了蛋白质组学方面的信息.     

猪链球菌7型强弱毒株SBP_bac_5差异表达蛋白的鉴定

前期研究表明细菌胞外溶质结合蛋白家族5 (SBP_ bac_5)基因在猪链球菌7型(S.suis 7)型强毒株WC0711中表达量高于弱毒株M13中,推测SBP_ bac_5为S.suis7型强弱毒株差异性表达蛋白.为进一步证实该结果,本研究采用real-time RT-PCR技术在基因水平检测S.suis7型强弱毒株中SBP bac_5 mRNA的表达量差异,结果显示,强毒株WC0711中SBP bac 5的mRNA表达量为在弱毒株M13中的4.59倍.将SBP bac 5的PCR产物克隆于pET-30a中,通过E.coli BL21诱导表达,纯化后的蛋白免疫新西兰白兔制备抗血清检测S.suis强弱病毒株中SBP bac 5的表达量差异.结果显示,重组蛋白获得高效表达,S.suis 7型强毒株WC0711中SBP bac 5表达量是弱毒株M13的3.8倍.     

猪链球菌2型05ZYH33菌株启动子的筛选和鉴定

为了筛选和鉴定用于猪链球菌(S.suis)致病因子和致病机制研究的启动子,本研究通过质谱鉴定了来自S.suis2型05ZYH33菌株的5个高丰度蛋白质,并通过其驱动GFP蛋白和甘露糖苷酶SSU05_1921的表达水平,评估了这5个蛋白相应的基因启动子的强弱。结果显示,这5种蛋白质分别为SSU05_0177、SSU05_0530、SSU05_1503、SSU05_1815和SSU05_1868,相应的5个启动子P0177、P0530、P1503、P1815和P1868均能够有效驱动GFP在E.coli和S.suis中的高水平表达,但是P1815、P0177和P1868不能驱动SSU05_1921在S.suis中的表达。P0530和P1503能够驱动SSU05_1921在S.suis中的表达,并且P1503启动子驱动GFP和SSU05_1921的表达水平均比P0530启动子驱动的高两倍以上。因此,P0530和P1503是S.suis的强启动子,并且P1503更强于P0530。这在高水平表达GFP用于标记菌体,以及构建无启动子基因的回复突变菌株中发挥关键作用,在S.suis遗传操作中具有很好的应用前景。     

猪链球菌2型安徽分离株毒力基因cps2J、mrp、epf、sly的检测及其序列分析

为了解19株猪链球菌2型(S.suis 2)安徽分离株的毒力基因型及毒力基因变异情况,通过PCR扩增S.suis 2毒力基因cps2J、mrp、epf和sly,对这4种毒力基因的扩增片段进行序列测定,并与国内外其他分离株的基因序列进行比较.结果显示,cps2J、mrp、epf和sly基因在19株S.suis 2中的检出率分别为100％、68.4％、68.4％、78.9％;19个受试菌株共分为7个毒力基因型,其中cps2J+/mrp+/epf+/sly+占57.9％,为优势基因型;S.suis 2安徽分离株4种毒力基因的检测序列之间及其与国内其他地区S.suis 2分离株的相应序列同源性均在99.1％以上,与国外S.suis 2分离株的相应序列同源性在87.8％～100％之间;19株cps2J+菌株中有1株菌cps2J基因序列有变异,13株mrp十菌株中有6株菌mrp基因序列发生变异,检测的epf和sly基因序列没有变异.表明cps2J +/mrp+ /epf+/sly+是S.suis 2安徽分离株优势毒力基因型,检测的S.suis 2安徽分离株cps2J、epf和sly基因部分序列较保守,mrp基因部分序列存在较大的变异.     

猪链球菌的分离鉴定及其致病性分子基础研究

为调查猪源链球菌(S.suis)血清型的流行特点、主要毒力基因的分布及致病力情况,本研究针对来自我国部分地区800份疑似S.suis发病猪的病料样品进行S.suis的分离鉴定、血清型分型、致病力试验及主要毒力基因型检测。研究结果显示,在800份样品中分离到41株S.suis(5.13%);其中,血清型2型或1/2型S.suis 10株(24.4%),3型6株(14.6%),8型3株(7.3%),9型7株(17.1%),21型1株(2.4%),未定型14株(34.1%);致病力试验结果显示,以1×108 cfu/mL S.suis菌液接种昆明小鼠,41个分离株中具有致死性的菌株19株,其中6株致死性较强,13株致死性较弱;毒力基因检测结果显示gdh、fbps、orf2、gapdh、sly、ef和mrp基因的检出率分别为100%、82.9%、75.6%、41.5%、39.0%、19.5%和12.2%,以gdh+orf2+fbps+gapdh型(9株)、gdh+orf2+fbps型(8株)和sly+gdh+orf2+fbps+gapdh型(6株)为主要的毒力基因型。本研究结果揭示了我国S.suis各血清型的流行情况,初步探明S.suis致病力的强弱,为深入开展S.suis防控技术研究及其致病机理奠定了基础。     

雏鸡法氏囊蛋白质组学双向电泳技术的建立及其初步分析

为了建立并优化鸡法氏囊蛋白质组学的双向电泳技术体系,以不同日龄雏鸡的法氏囊组织为研究对象,用固相pH梯度胶条进行等电聚焦、SDS-PAGE垂直电泳,采用不同的样品制备方法,对上样量、水化、等电聚焦、胶条平衡和凝胶染色方法等进行一系列优化,并应用PDQuest8.0.1软件对图谱进行初步分析.结果显示法氏囊组织在pH 5～8范围、17 cm的2-DE胶上可以得到很好的分离,胶体考染后经PDQuest软件分析,在正常法氏囊组织可检测到800个以上蛋白点,不同2-DE图谱间蛋白点平均匹配率为83.5%,不同日龄雏鸡法氏囊存在有明显表达差异的蛋白质点37个,其中表达上调蛋白点17个,表达下调蛋白点11个,新增蛋白点5个,消失蛋白点4个,试验建立的鸡法氏囊组织蛋白质组双向电泳技术为法氏囊发育进化及其免疫功能的研究提供了新技术和方法.     

嗜水气单胞菌胞外产物的生物活性及主要蛋白型分析

在血清学分型的基础上制备了23 株嗜水气单胞菌和1 株温和气单胞菌的胞外产物。分析这些菌株的溶血活性、蛋白酶解活性和细胞毒活性。结果表明,上述3 种生物学活性与菌株血清型间没有严格的对应关系。SDS PAGE分析显示,胞外产物的SDS PAGE图谱主要包括35 ku,45 ku,53 ku 3个主要蛋白质条带。有别于此前的多数报道,35 ku的蛋白质条带为多数菌株(22/24)所共有。根据凝胶上主要蛋白条带在不同菌株中的分布,可初步将其中的18 株细菌分为3 个胞外产物ECPs蛋白型,该ECPs蛋白型显示与血清型有一定的对应关系。经蛋白酶K消化处理的ECPs,银染可见脂多糖(LPSs)的典型O 糖侧链结构,显示脂多糖是组成粗制ECPs的重要组分之一。     

牛布鲁菌强毒株544与猪型疫苗株S2基因组差异筛选与鉴定

应用代表性差异分析技术(Representational difference analysis,RDA)对流产布鲁菌强毒株544和疫苗株S2进行基因组差异分析。经过3轮差减杂交,对差异片段进行Southern-blot验证,BLAST分析和PCR鉴定,最终获得疫苗株S2特有的3条差异片段与已测序的所有猪布鲁菌(Brucella suis)同源性均为100%,只在弱毒株S2基因组中检测出,而在强毒株544中不存在,为建立布鲁菌自然感染菌与疫苗株的基因鉴别诊断方法奠定了基础。     

水牛卵泡液差异蛋白质双向电泳方法的建立及质谱分析

旨在研究沼泽型水牛成熟前后卵泡内差异表达蛋白质的变化规律。采用双向凝胶电泳技术分离成熟卵泡液和未成熟卵泡液总蛋白质,建立和优化了卵泡液的双向电泳体系,并使用质谱鉴定差异表达蛋白点。结果显示,丙酮沉淀法处理得到的总蛋白质样品后,在24cm(pH 4～7)胶条且上样量350μg时得到分辨率较好的双向电泳图谱。软件分析得到11个差异蛋白点,以成熟卵泡液作为对照,5个蛋白点表达上调,3个蛋白点表达下调,1个蛋白点缺失,2个蛋白点在未成熟卵泡液中特异性表达。质谱成功鉴定出4个蛋白质:过氧化物酶-2、醛糖还原酶、牛纤维蛋白原的晶体结构、转甲状腺素蛋白。该研究建立了良好的卵泡液双向电泳体系,分析并鉴定一批水牛卵泡液差异蛋白质,对于研究水牛卵母细胞的发育微环境和成熟机制提供了新的研究线索。     

4株鸭源肠球菌的鉴定和致病性

对临床分离的4株鸭源肠球菌郑1株、郑2株、郑3株、北京株和1株粪肠球菌参考菌株进行了系统鉴定,并用SDS-PAGE和Western-blot技术对各菌株细胞壁蛋白图谱进行比较分析。结果5个菌株的形态、染色、生理生化特性均与粪肠球菌特性一致;它们均对青霉素、万古霉素和庆大霉素敏感而对四环素耐药;5个菌株人工感染雏鸭及小白鼠均有致病性,但各菌株间致病力存在差异,北京株最强,参考株最弱,其余3株介于北京株和参考株之间;各菌株的细胞壁蛋白经SDS-PAGE在相对分子质量33 370~131 690之间均显示数十条蛋白带,其中郑2株和北京株在相对分子质量66 840处均有1条染色较深的蛋白带,而用Western-blot分析显示抗北京株胞壁蛋白抗体只能检测到北京株相对分子质量为66 840的抗原蛋白。以上结果表明,这5个被检菌株为致病性粪肠球菌,且致病性以北京株最强。     

Experimental infections of mice and pigs with Streptococcus suis type 2.

Five inbred strains of mice were tested for their susceptibility to Streptococcus suis type 2 including the type strain, two isolates from meningitis in pigs and two isolates from tonsils of clinically healthy pigs. C57BL/6, ICR and ddY strain mice showed lower susceptibility to all strains of S. suis type 2 than BALB/c and SS strain mice. The type strain and the isolates from diseased pigs produced septicaemia and meningitis in BALB/c and SS mice inoculated with 10(8) colony forming unit of the bacteria and 60 to 100% of these infected mice died. On the other hand, mice inoculated with the isolates from healthy pigs showed mild clinical signs but none of them died. In BALB/c mice which died or developed nervous signs, the purulent meningo-encephalitis, myocarditis, ophthalmitis, labyrinthitis and otitis media were observed. S. suis type 2 antigen was demonstrated in these lesions by immunoperoxidase staining using rabbit S. suis type 2 antiserum. These results were similar to those in the experimentally infected pigs with these virulent and avirulent strains against mice. These results indicate that BALB/c and SS strains of mice are useful as an experimental model of S. suis type 2 infections in pigs, and that there are virulent and avirulent strains against mice and pigs among the strains of S. suis type 2.     

Identification and distribution of putative virulent genes in strains of Streptococcus suis serotype 2

In order to identify gene sequences unique to the virulent strains, suppression subtractive hybridization (SSH) was conducted using virulent Streptococcus suis type 2 (SS2) strain HA9801 and avirulent S. suis type 2 strain T15. Thirty genomic regions were absent in T15, and the DNA sequences of these regions in HA9801 were determined. These DNA fragments, containing putative virulence genes, encoded 28 proteins that were homologous to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems and others of unknown function. According to the published SS2 genomic sequence of the Chinese strain 98HAH33, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, groups and times. The results showed that these 14 DNA fragments were widely distributed in 37 detected SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution of the fragments.     

Immunization of mice against Streptococcus suis serotype 2 infections using a live avirulent strain.

In this study, the IgG response of mice injected with two virulent strains and one avirulent Streptococcus suis capsular type 2 strain was compared by Western blotting. The serum from mice immunized against the avirulent strain could recognize most proteins of the various strains tested and similar results were obtained with serum from mice injected with virulent strains. The live avirulent strain was injected twice (days 0 and 10) to groups of five mice, and four virulent strains from different geographical origins were used to challenge the animals. All mice, except one in one group, survived the challenge. These results suggest that a live avirulent strain could be used for immunization of swine, the natural host.     

家蚕对浓核病毒(镇江株)抵抗性和感受性品种的中肠组织蛋白比较分析

为了探明家蚕对浓核病毒(镇江株)抵抗性和感受性品种间在分子机制上的差异,通过蛋白质双向电泳(2-DE)和基质辅助质量飞行时间质谱技术(MALDI-TOF MS)对两个不同抗性家蚕品种的中肠组织蛋白进行了比较分析。2-DE电泳结果表明,两个品种间的中肠组织蛋白斑点数及位置、形态等差异很小,进一步比较获得了9个差异蛋白斑点,其中感受性蚕品种JS有6个,抵抗性蚕品种NIL有3个。经MALDI-TOF MS鉴定结果显示,感受性品种JS有4个差异蛋白斑点可能分别为氢离子转运ATP合酶β亚基1、氢离子转运ATP合酶β亚基2、组织蛋白酶D或3-羟酰辅酶A脱氢酶;抵抗性品种NIL中有2个差异蛋白点可能是组织蛋白酶。     

A comparison of virulent and avirulent strains of Salmonella choleraesuis and their ability to invade Vero cell monolayers.

The mechanisms of invasion used by virulent and avirulent Salmonella choleraesuis were compared using a Vero cell invasion assay. Mouse virulent S. choleraesuis strain 38 and avirulent strain 9 were examined for their ability to invade and survive in Vero cells. The assay was performed by S. choleraesuis infection of the Vero cell monolayer alone and in the presence of various treatments applied to the Vero cell monolayers. Intracellular S. choleraesuis colony forming units were then counted to characterize the mechanism of bacterial uptake. Invasion was not affected by colchicine, but was significantly inhibited in the presence of cytochalasins B and D, chloroquine, and dansylcadaverine. Inhibition by the above substances suggested the importance of microfilaments and of receptor recycling in receptor mediated endocytosis. Both bacterial strains had decreased invasion in the presence of mannose and after enzymic treatment with trypsin. Mannose exposure caused a significant 48% decrease in the uptake of virulent S. choleraesuis 38 and a 28% decrease in avirulent S. choleraesuis 9. Inhibition of endosome acidification did not affect the virulent strain 38 as much as it affected avirulent strain 9. Results from these experiments suggested that Vero cell invasion by S. choleraesuis was due to host uptake by receptor mediated endocytosis, and was mediated in part by mannosesensitive adhesins. Outer membrane proteins were extracted from the virulent and avirulent strain and compared using SDS-PAGE following surface protein labeling with ¹²⁵I. Virulent S. choleraesuis 38 had a unique 35 kD protein. The outer membrane proteins of both strains were then examined by radio-immunoprecipitation and western blot using guinea pig polyclonal antisera and the 35 kD protein was again found to be unique to the virulent strain 38. Antisera against the 35 kD protein significantly inhibited invasion of Vero cells by S. choleraesuis strain 38.     

Staphylococcus hyicus virulence in relation to exudative epidermitis in pigs.

Staphylococcus hyicus strains with different phage types, plasmid profiles, and antibiotic resistance patterns were isolated from piglets with exudative epidermitis. The strains could be divided into virulent strains, producing exudative epidermitis, and avirulent strains, producing no dermal changes when injected in experimental piglets. The results showed that both virulent and avirulent strains were present simultaneously on diseased piglets. This constitutes a diagnostic problem. Concentrated culture supernatants from nine virulent strains injected in the skin of healthy piglets produced a crusting reaction in all piglets. Acanthosis was observed in the histopathological examination of the crustaceous skin. Concentrated culture supernatants from nine avirulent strains produced no macroscopic or microscopic skin changes. Protein profiles from all virulent strains and seven out of nine avirulent strains showed a high degree of protein band homology. An approximately 30 kDa protein present in all concentrated culture supernatants capable of producing skin changes, could not be detected in samples that did not produce skin changes. No other protein showed a similar association. It is concluded that crusting reaction of piglet skin is a suitable indicator of virulence in S. hyicus in relation to exudative epidermitis, and that virulent strains produce a 30 kDa protein, absent in concentrated culture supernatants from avirulent strains. This 30 kDa protein might be an exfoliative toxin.     

猪多杀性巴氏杆菌对HeLa细胞附着能力的研究

本研究通过猪肺疫的活菌疫苗和死菌疫苗多杀性巴氏杆菌菌株(Pasteurella multocida,Pm)对小鼠的毒力试验测定它们的毒力性。结果表明,死菌疫苗Pm的毒力性比活菌疫苗Pm的强,即死亡率分别为10 0 %和0 %。通过两菌株对He L a细胞的附着试验测定它们的附着能力,结果证明强毒菌的附着能力明显地比弱毒菌强(P<0 .0 1) ,平均附着数分别为11.96和2 .4 4 ;从上述菌株细胞荚膜中分别提取荚膜蛋白,用SDS- PAGE分离测定两菌株荚膜蛋白质结构,结果表明39k Da荚膜蛋白是强毒菌的特异性蛋白。以上研究结果证明Pm的毒力与He L a细胞的附着能力是密切相关的,同时暗示本菌39k Da荚膜蛋白可能与它们的毒力和He L a细胞的附着能力有关     

新城疫病毒人工感染鹅脾脏差异表达蛋白质组初步分析

脾脏是新城疫病毒(Newcastle disease virus,NDV)感染的重要靶器官,本试验旨在从分子水平上分析NDV与宿主之间的相互作用,探寻早期基因Ⅳ型强毒Herts/33和晚期基因Ⅶ型强毒JS5/05造成鹅脾脏病变差异的相关蛋白。30日龄非免疫鹅分别人工感染NDV强毒株Herts/33和JS5/05,并于感染后36、72、108h采集2个感染组和对照组的鹅脾脏,提取脾脏蛋白,以17cm、pH5～8的IPG胶条进行二维电泳,运用PDQuest 8.0.1软件对凝胶图谱进行差异蛋白分析。结果显示:与对照组相比,Herts/33感染组和JS5/05感染组脾脏组织分别有154个和148个蛋白出现了显著的差异表达,其中有86个蛋白点是不同感染组共有的差异点,包括52个感染后上调表达蛋白点,34个下调表达蛋白点;另外,有130个差异蛋白点为NDV感染鹅后不同毒株之间产生的差异表达,包括71个感染后上调表达蛋白点,59个下调表达蛋白点。基因Ⅳ型NDV强毒和基因Ⅶd亚型NDV强毒分别感染鹅后,能引起宿主脾脏组织蛋白表达谱发生不同的改变,这为进一步研究Ⅶd亚型NDV对水禽致病性增强的机制提供了重要线索。     

家蚕绵茧突变品系中部丝腺的差异蛋白组学分析

为了获取与家蚕绵茧突变形成相关蛋白质的基础信息,采用蛋白质双向电泳技术对结茧性状具有明显差异的正常茧、绵茧、丝胶茧3个家蚕品系5龄期幼虫中部丝腺不同区段的蛋白质进行双向电泳(2-DE)分析,图谱中的蛋白点主要集中在分子质量14～70 kD、等电点(pI)4～9的区域。采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对部分表达量差异显著的蛋白点进行鉴定,获得35种可信的蛋白质,绵茧突变品系与正常茧、丝胶茧品系相比表达量有显著差异的蛋白质包括参与能量代谢、蛋白质合成等生物学过程的功能蛋白质,其中丝氨酸蛋白酶抑制剂16(serpin16)和丝氨酸蛋白酶抑制剂18(serpin18)可能与家蚕绵茧突变形成相关。     

Studies on Erysipelothrix insidiosa S. rhusiopathiae. 1. Morphology, cultural features, biochemical reactions and virulence

Sixty-two E. insidiosa strains isolated from joints or regional lymph nodes of pigs were examined from the point of view of morphology, cultural aspects, biochemical activity and virulence. All the strains consisted of gram-positive, short rods, which were similar on solid and fluid media. All strains formed H₂S. Otherwise the biochemical activity was rather low except in 1 strain (no. 18), which was very active. One strain (no. 36) was rather inactive, since it showed no other activity than H₂S formation. This latter strain was the only one that was avirulent for mice. The rest of the strains (61) were strongly virulent for mice (LD50 0.5 × 10^−4.17 to 0.5 × 10^−8.5).Of 7 strains examined for virulence for pigs by intracutaneous injection of 0.1 ml broth culture, 6 were virulent. The 7th, which was avirulent, was the one that was also avirulent for mice.